Nucleic acid amplification techniques provide powerful tools for the study of genetic material. The polymerase chain reaction (PCR) is one of the more frequently utilized techniques and has applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery and criminal forensics, among others.
For many applications, in addition to amplifying a target nucleic acid sequence, it may be useful to further characterize the sequence by treatment with a nucleic acid hybridization probe. An example of a probe would be a labeled single stranded polynucleotide which is complementary to all or part of the target sequence. Probe hybridization may provide additional sequence selectivity over simple PCR amplification as well as allowing for the characterization of multiple sequence sites within the target nucleic acid sequence in an independent manner.
Traditionally, PCR and probe hybridization processes have been performed as separate reactions. More recently, the separate reactions have been integrated into a single reaction using a single reagent mixture containing both nucleic acid amplification reagents and hybridization reagents. Some of the many advantages of combining the reactions include reducing the reagent addition steps and utilizing fewer reagents.
Methods for the simultaneous analysis of multiple genes are in ever growing demand. Microarrays are an ideal platform for such analysis in scientific, clinical and environmental contexts, since their miniature size allows one to arrange up to hundreds or thousands of biological probes in a relatively small space and reaction volume. A microarray is a collection of microscopic DNA spots attached to a solid surface, forming an array for the purpose of expression profiling (monitoring expression levels for thousands of genes simultaneously). The combined nucleic acid amplification and hybridization reaction can be utilized with microarray applications for optimal analysis.
Currently, buffers used to host the combined reaction facilitate higher reaction temperatures, which cause expansion and the potential for leakage in reaction vessels. The higher temperature reaction may also create bubbles that interfere with the recognition of hybridization signals.